The ability to introduce manipulated genes and delete or modify endogenous genes in the germline of animals is a major technological advance in biology, enabling investigators to answer questions about gene function which must be analyzed in a whole animal model system. Transgenic and knockout animals have been instrumental in providing insights into mechanisms of developmental gene regulation, cellular interactions within the immune system, and the effect of oncogenes on growth and differentiation. Demand for these services has grown significantly since the last review, approximately doubling every two years. A new Facility Manager was recruited in early 2000 with a specific expertise in generating knockout mice. In calendar year 1999, there were 21 requests for services. In 2003, 84 requests were completed (76 DNA Constructs Injected, 8 ES Cell Lines Injected). The number of peer-reviewed funded users has increased from nine at the last review to 15 in calendar year 2003. The use from these 15 peer-reviewed funded investigators represents six different Research Programs and all three Divisions of the Fox Chase Cancer Center (FCCC). Ninety-nine percent (99%) of Facility usage is by peer-reviewed funded investigators. As described in the last application, we planned to add needed cryopreservation services. Cryopreservation has been established and has become a routine service. It is currently used by 11 investigators. A total of 88 lines have been preserved to date. The availability of this service has resulted in several clear benefits, including the ability to bank mouse lines for future study, to relieve pressure on existing mouse colony space, to provide a means for propagating reproductively incompetent mouse lines, and to provide insurance against the loss of irreplaceable mouse lines should a catastrophic event occur in the mouse colony. The previous review noted a need to provide cryopreservation and gene knockout services as well as to increase Facility usage. All three points have been fully addressed. Both cryopreservation and gene targeting are now accomplished routinely, with a 100% success rate. Productivity and usage have increased by 200% and 100%, respectively, since 1998.